Electrochemical detection nanostructure, systems and uses thereof

Abstract

Described herein are DNA-nanostructures that can be used in an assay to detect and/or quantify an analyte of interest. Aspects of the DNA-nanostructure can include a single DNA molecule composed of hairpin structural motifs, an anchor recognition moiety, and a signal moiety, where the anchor recognition moiety and the signal moiety are in effective proximity to each other such that the tethered diffusion of the signal molecule can be altered based upon binding status of the anchor recognition moiety. Also described herein are methods of making and using the DNA-nanostructures.

What is claimed is: 
     
         1 . A method for detecting an analyte, the method comprising:
 contacting a sample containing the analyte to a DNA nanostructure coupled to a surface of an electrode, wherein the DNA nanostructure comprises:
 a single continuous DNA molecule comprising: a first hairpin structural motif, a second hairpin structural motif, a first segment of single stranded DNA, and a second segment of single stranded DNA, 
 wherein the first hairpin structural motif and the second hairpin structural motif are attached to each other via the first segment of single stranded DNA, wherein the second segment of single stranded DNA is attached to the second hairpin structural motif such that the second segment of single stranded DNA forms a single stranded tether region at one end of the single continuous DNA molecule; 
 an anchor recognition moiety, wherein the anchor recognition moiety is covalently coupled to a region of the single continuous DNA molecule between the first hairpin structural motif and the second hairpin structural motif; and 
 a signal moiety coupled to an end of the single continuous DNA molecule, 
 wherein a terminal base of the second segment of single stranded DNA is coupled to the surface of the electrode; and 
   detecting a change in a tethered diffusion of the signal moiety relative to the electrode surface when the analyte binds to the anchor recognition moiety.   
     
     
         2 . The method of  claim 1 , wherein detecting the change in a tethered diffusion comprises detecting a change in an electrochemical current at the surface. 
     
     
         3 . The method of  claim 1 , further comprising binding the analyte to the anchor recognition molecule. 
     
     
         4 . The method of  claim 1 , further comprising rinsing to remove unbound analyte from the anchor recognition molecule. 
     
     
         5 . The method of  claim 1 , further comprising assembling the DNA nanostructure on the electrode. 
     
     
         6 . The method of  claim 5 , wherein assembling the DNA nanostructure on the electrode comprises immobilizing a thio-DNA comprising the second hairpin structural motif and the second segment of single stranded DNA to the surface of the electrode, ligating an anchor recognition unit comprising the first segment of single stranded DNA and the first hairpin structural motif, so that the second segment of single stranded DNA is attached to the second hairpin structural motif, and ligating a third segment of single stranded DNA coupled to the signal moiety to the anchor recognition unit. 
     
     
         7 . The method of  claim 1 , wherein the DNA nanostructure further comprises a linker having a reactive group capable of attaching to the surface of the electrode, wherein the linker is attached to the terminal base of the second segment of single stranded DNA. 
     
     
         8 . The method of  claim 7 , wherein the reactive group is selected from the group consisting of: a carboxyl group, amino group, aromatic amine group, a chloromethyl group, an amide group, a hydrazide group, a hydroxyl group, a thiol group, an epoxy group, and combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein the single continuous DNA molecule has a sequence that is 1-100% identical to one of SEQ ID NOs: 7-8. 
     
     
         10 . The method of  claim 1 , wherein the signal moiety is a redox molecule. 
     
     
         11 . The method of  claim 1 , wherein the signal moiety is methylene blue. 
     
     
         12 . A method for detecting an analyte, the method comprising:
 contacting a sample containing the analyte to a DNA nanostructure coupled to a surface of an electrode, wherein the DNA nanostructure comprises:
 a single continuous DNA molecule comprising: a first hairpin structural motif, a second hairpin structural motif, a first segment of single stranded DNA, and a second segment of single stranded DNA, wherein the first hairpin structural motif and the second hairpin structural motif are attached to each other via the first segment of single stranded DNA, wherein the second segment of single stranded DNA is attached to the second hairpin structural motif such that the second segment of single stranded DNA forms a single-stranded tether region at one end of the single continuous DNA molecule; 
 an anchor recognition moiety, wherein the anchor recognition moiety is covalently coupled to the single continuous DNA molecule and extends from the single continuous DNA molecule; and 
 a signal moiety coupled to an end of the single continuous DNA molecule opposite from the tether region, 
 wherein a terminal base of the second segment of single stranded DNA is coupled to the surface of the electrode; and 
   detecting a change in a tethered diffusion of the signal moiety relative to the electrode surface when the analyte binds to the anchor recognition moiety.   
     
     
         13 . The method of  claim 12 , wherein detecting the change in a tethered diffusion comprises detecting a change in an electrochemical current at the surface. 
     
     
         14 . The method of  claim 12 , further comprising binding the analyte to the anchor recognition molecule. 
     
     
         15 . The method of  claim 12 , further comprising rinsing to remove unbound analyte from the anchor recognition molecule. 
     
     
         16 . The method of  claim 12 , further comprising assembling the DNA nanostructure on the electrode. 
     
     
         17 . The method of  claim 12 , wherein the DNA nanostructure further comprises a linker having a reactive group capable of attaching to the surface of the electrode, wherein the linker is attached to the terminal base of the second segment of single stranded DNA. 
     
     
         18 . The method of  claim 17 , wherein the reactive group is selected from the group consisting of: a carboxyl group, amino group, aromatic amine group, a chloromethyl group, an amide group, a hydrazide group, a hydroxyl group, a thiol group, an epoxy group, and combinations thereof. 
     
     
         19 . The method of  claim 12 , wherein the signal moiety is a redox molecule. 
     
     
         20 . A method for detecting an analyte, the method comprising:
 contacting a sample containing the analyte to a DNA nanostructure coupled to a surface of an electrode, wherein the DNA nanostructure comprises:
 a single continuous DNA molecule comprising: a first hairpin structural motif, a second hairpin structural motif, a first segment of single stranded DNA, and a second segment of single stranded DNA, 
 wherein the first hairpin structural motif and the second hairpin structural motif are attached to each other via the first segment of single stranded DNA, wherein the second segment of single stranded DNA is attached to the second hairpin structural motif such that the second segment of single stranded DNA forms a single-stranded tether region at one end of the single continuous DNA molecule; 
 an anchor recognition moiety, wherein the anchor recognition moiety is covalently coupled to the single continuous DNA molecule and extends from the single continuous DNA molecule; and 
 a signal moiety, wherein the signal moiety is coupled to an end of the single continuous DNA molecule opposite from the tether region, 
 wherein the signal moiety is in effective proximity to the anchor recognition moiety so that binding of an analyte to the anchor recognition moiety changes a tethered diffusion of the signal moiety; and 
 wherein the DNA nanostructure is coupled to the surface at a terminal base of the second segment of single stranded DNA so that changes in the tethered diffusion of the signal moiety changes an electrochemical current at the surface; and 
   detecting a change in a tethered diffusion of the signal moiety relative to the electrode surface when the analyte binds to the anchor recognition moiety.